Aspergillus fumigatus is the most common cause of invasive mold infections. Even with advances in therapy and early diagnostics, mortality rates remain high. Plasmacytoid dendritic cells (pDCs) comprise a functionally distinct lineage of DCs that rapidly produce copious amounts of type I interferons (IFNs) upon stimulation with viruses, predominantly via mechanisms dependent on sensing of viral nucleic acids. pDCs link innate to adaptive immunity by secreting cytokines and by priming T cells. The interplay between pDCs and fungi has not been well characterized. The application builds upon compelling preliminary data demonstrating that: 1) human pDCs directly inhibit fungal growth via a mechanism that involves A. fumigatus-induced pDC death; 2) following stimulation with A. fumigatus hyphae, pDCs release cytokines, including type I IFNs; 3) depletion of pDCs renders mice hypersusceptible to pulmonary and intravenous challenge with A. fumigatus by a mechanism that appears to be due, at least in part, to a dysregulated immune response; and 4) pulmonary infection with A. fumigatus results in pDC influx into the lungs. We hypothesize that pDCs play a major role in host defenses against invasive aspergillosis by mediating direct antifungal activity and by modulating the innate and adaptive immune response. To address this hypothesis, we will define the contribution of pDCs in the defense against A. fumigatus using both in vitro and in vivo models. In Aim 1, we will explore the mechanistic basis of our observations that incubation of pDCs with A. fumigatus in vitro results in fungal recognition, antifungal activity and cytokine release. The receptors required for pDC recognition of the conidial and hyphal fungal morphotypes will be explored. The mechanism of pDC death induced by A. fumigatus will be elucidated. How pDCs mediate antifungal activity will be characterized using both intact pDCs and pDC lysates. Finally, the cytokine and chemokine response of pDCs to A. fumigatus will be investigated. In aim 2, we will follow up our demonstration that pDCs have a non- redundant role in host defenses against aspergillosis by illuminating the mechanisms by which this occurs. The effect of pDC depletion on mortality, fungal burden, immune cell recruitment, pathology and cytokine response will be determined in murine models of invasive aspergillosis. The arms of the immune system required for pDC-mediated protection will be examined by co-depleting pDCs and specific leukocyte subsets and with knockout mice. Lastly, we will determine whether pDCs associate with conidia and hyphae in vivo using flow cytometry and microscopy. Completion of these studies over the funding period will result in major conceptual advances in fungal pathogenesis, fungal immunology and pDC biology. Moreover, the knowledge gained may lead to novel strategies to prevent and treat invasive mycoses.